This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal concerns the study of: i) the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT) and AlkB, from both E. coli and human;ii) a drug-resistant regulator MgrA in Staphylococcus aureus. AGT and AlkB repair the alkylation DNA base damage via direct removal of the damage. The repair activity of AGT is an important component for cellular resistance to the toxic and mutagenic effects of alkylation damage since the deleterious modification that is corrected by this family of proteins, O6-alkylguanine, represents one of the most mutagenic lesions known. We have recently solved the crystal structure of the human AGT/DNA complex with hAGT recognizing a base damage mimic. We plan to apply a chemical-crosslinking technique to stabilize AGT on normal DNA without damage. We hope to capture the structure of the non-specific DNA damage-searching mode of the repair protein. We are also working on structural studies of a very important group of DNA repair proteins, AlkB. The function of these proteins has only identified recently and this is an emerging frontier in cancer research. We are working on crystal structures of both E. coli and human AlkB proteins. Crystals of the human protein have been obtained in our group. We also work on structural studies of MgrA, a global regulator and major virulence determinant in S. aureus. Staphylococcus aureus is an important human pathogen responsible for most wound and hospital acquired infections. We demonstrate here that MgrA is a major virulence determinant during infection and is a regulator of antibiotic resistance in S. aureus. We have solved the first crystal structure of apo-MgrA at 2.8 [unreadable] resolution, which provides a template for designing potential therapeutic interventions for staphylococcal infections. We have obtained crystals of MgrA/DNA complexes and are also working on drug-bound MgrA structures. These works will provide insight into the regulatory mechanism of this very important protein.